Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) consist of a genetically diverse and distinctive grouping of water-soluble hydrolytic enzymes that typically act on the ester bonds of lipid substrates. Lipids can include fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, fatty acyls, polyketides, and fatty acids, and exist as a number of variations containing different additional chemical structures such as phospholipids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, etc. Lipases have been used in ester and amide synthesis, kinetic resolutions or asymmetric synthesis to obtain optically pure compounds, and lipid modifications (Bomscheuer and Kazlauskas, 2005). Lipases play an essential role in: (1) the metabolism of dietary lipids, (2) injury and inflammation, (3) cell biology, (4) fermentation, (5) biocatalysis, (6) vitamin absorption, (7) laundering, (8) synthesis of pharmaceuticals and many other biological and chemical processes. Such wide and varying roles have been attributed to lipase stability in organic solvents, high specificity, high enantio-selectivity and regio-selectivity, and a general lack of a need of cofactors for their action. Genes encoding lipases have been found in most, if not all, types of organisms.
Typically, the tertiary structure of lipases includes the alpha/beta (α/β) hydrolase fold pattern (Ollis et al., 1992), also common in peptidases and esterases (Holmquist, 2000), and can be composed of a core of up to eight beta strands, connected and surrounded by alpha helices. The active sites of lipases are usually formed by at least a catalytic triad consisting of a serine residue as the nucleophile, a histidine residue, and an aspartic or glutamic acid residue. The active site residues are located in a hydrophobic pocket that is covered by a flap or lid structure, usually composed of amphiphilic α helices (Anthonsen et al., 1995).
Lipases typically act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. There are typically four basic steps in lipase hydrolysis and/or alcoholysis (i.e., ethanolysis), which involves a conformational change of the lipase itself. First, the lipase is adsorbed and activated by the opening of the hydrophobic pocket by displacement of the lid structure, by the so-called interfacial activation. Once the pocket is opened, the ester bond of the lipid substrate is able to reach and bind to the lipase active site. Second, the nucleophilic oxygen of the serine side chain binds the carbonyl carbon of the ester bond, forming a tetrahedral intermediate, stabilized by hydrogen bonding with amide nitrogen atoms of the amino acid residues nearby. Third, the ester bond is cleaved, which frees an alcohol and produces an acyl-enzyme complex. Last, the acyl-enzyme is hydrolyzed upon entry of a water molecule or alcohol into the active site. This frees the fatty acid (in case of water as nucleophile) or ester (in case of an alcohol as nucleophile) and the lipase is regenerated.
Due, in part, to their diverse functioning and structure, as revealed by sequence analysis and crystallography, lipases belong to different enzyme subclasses or families. Pseudozyma (formerly Candida) antarctica, is a basidiomycetous yeast strain isolated from Lake Vanda in Antarctica that produces two differently functioning lipases: lipase A (CAL-A) and lipase B (CAL-B) (Ericsson et al., 2008). These two lipases have been previously characterized and the amino acid and DNA sequences encoding these lipases have been determined (Novo Nordisk A/S, by Hoegh et al., 1995). CAL-B is a widely used enzyme in organic synthesis on both the laboratory and commercial scale, especially in the resolution of racemic mixtures.
CAL-A is one representative of a new class of lipases and, due to its properties, including thermostability, has been used as a catalyst in the paper, wax, food, flavor, and biopharmaceutical industries. CAL-A has an unusual lid structure and C-terminal flap, which can accept very bulky substrates like highly branched acyl groups and sterically hindered alcohols and amines (Kirk and Christensen, 2002; Krishna et al., 2002; Schmidt et al., 2005). CAL-A also shows a higher homology to peptidase structures rather than typical lipase structures (Ericsson et al., 2008).
Mono- or poly-unsaturated fats with trans-isomer fatty acid(s) are commonly called “trans fats.” Trans-isomers contain chains where the carbon atoms next to the double bond are located geometrically opposite, whereas in cis-isomers the carbon atoms next to the double bond are geometrically on the same side. In the cis configuration, the naturally occurring unsaturated fatty acids have lower melting points than those of saturated fatty acids, and thus are found in liquid form. Typically, trans-fatty acids are found in food products as a result of a partial hydrogenation process. Trans-fatty acids have higher melting points than those of the cis-unsaturated fatty acids and are less susceptible to auto-oxidation and so can form a more stable solid or semi-solid fat. Dietary intake of trans-fatty acids has been linked to an increased risk for heart disease, diabetes, obesity, metabolic syndrome, Alzheimer's disease, cancer, liver dysfunction and infertility. For these reasons, attempts have been made to reduce the trans-fatty acid content in dietary products (Ratnayake and Cruz-Hernandez, 2009).
Lipases have gained significant commercial importance; however, the expression levels in native organisms are too low to meet these increasing needs. Therefore, numerous attempts have been made to optimize the activity, selectivity, sensitivity and stability of lipases. These include immobilizing the lipase on solid supports and using non-aqueous solvents as well as recombinant DNA techniques and protein engineering. Understanding the mechanisms underlying gene expression, protein folding and excretion of lipases enables higher-level production of these biocatalysts (Napolitano and Giuffrida, 2009).
Numerous lipase assay methods have been used to determine lipase activity, including, but not limited to, using colored or fluorescent substrates, which allow spectroscopic and fluorimetric detection of lipase activity, chromatography techniques including high-performance liquid chromatography (HPLC), silver ion chromatography, gas chromatography and thin layer chromatography, titration of fatty acids released from the substrate, mass spectrometry and controlled surface pressure or oil drop tensiometry.
Due to the central importance of lipase function in lipid metabolism and transport, and its implication in serious diseases and conditions such as heart disease, diabetes, obesity, metabolic syndrome, Alzheimer's disease, cancer, liver dysfunction and infertility, it is imperative to know not only how lipases work, but also how to improve the activity, selectivity, sensitivity and stability of lipases. What is desirable, therefore, are compositions and methods for producing a novel lipase variant, increasing the preference of a lipase for long fatty acid chains, increasing the range and number of fatty acid chains that a lipase is able to catalyze, and increasing the trans-selectivity of lipases and reducing or eliminating trans-fatty acids from lipid substrates. Such compositions and methods find particular utility in a variety of analytical assays and dietary regimens.